1. Field of the Invention
The present invention relates to site-specific heterobifunctional crosslinking reagents, biomolecules or bioaffecting molecules to which the present crosslinking reagents are bound, and kits containing biomolecules or bioaffecting molecules to which the present crosslinking reagents are bound.
2. Discussion of the Background
Heterobifunctional crosslinking reagents are widely used for linking effector molecules to biomolecules such as glycoproteins including antibodies, lectins, enzymes, and response modifiers.
In particular, immunoconjugates result from the chemical coupling of monoclonal antibodies with various effector molecules, which may include toxins (e.g., ricin A chain; reviewed by Moller, in, Immunol. Rev., p. 62, Copenhagen (1982)), biological response modifiers (e.g., cobra venom factor; reviewed by Vogel in Immunoconjugates. Antibody Conjugates in Radioimaging and Therapy of Cancer, pp. 170-188 Oxford Univ. Press 1987)), and low molecular weight drugs (e.g., doxorubicin; reviewed by Sela and Hurwitz in Immunoconjugates. Antibody Conjugates in Radioimaging and Therapy of Cancer, pp. 189-216, Oxford Univ. Press (1987)). The potential of such immunoconjugates for the development of novel anti-cancer therapeutics and imaging techniques has been studied by many investigators and has been reviewed by C.-W. Vogel in Immunoconjugates, Oxford Press (1987),
Several methods for coupling effector molecules to monoclonal antibodies are known. Currently used methods for the synthesis of immunoconjugates employ heterobifunctional crosslinking reagents which contain one amino-reactive residue (e.g., succinimidyl ester) and one sulfhydryl-reactive residue (e.g., pyridyl disulfide). An example of such heterobifunctional reagents is N-succinimidyl-3-(2-pyridylthio)propionate (SPDP) which is described in Biochem. J., vol. 173, pp. 723-737 (1978). Upon incubation of antibodies with SPDP, the crosslinking reagent is coupled via an amide bound to one of the primary amino groups of the antibody, thereby introducing a pyridyl disulfide moiety to which a sulfhydryl-containing effector molecule can be coupled.
One major problem of this technique, however, is the random distribution of amino groups throughout the entire antibody molecule including the antigen binding region. Thus, the crosslinking agents may bind to the antibody at a site close to the binding region of the antibody and, thus, interfere with the binding of the antibody. Therefore, use of the currently available heterobifunctional crosslinking agents diminishes the antigen binding capability of the resulting immunoconjugates and, thereby, limits their efficacy as therapeutic and diagnostic agents.
More recently, the reaction of amine-containing molecules with the carbohydrate region of antibodies has been investigated for the purpose of preparing antibody derivatives, see D. J. O'Shannessy, Int. Soc. Biorecognition Tech., vol. 3, pp. 4-6 (1988).
PCT Patent Application W087/06837 discloses linking amine-derivatives of folic acid to antibodies which contain an oxidized carbohydrate moiety.
U.S. Pat. No. 4,671,958 discloses the use of crosslinkers, one end of which contains a hydrazine derivative, for the purpose of reacting with oxidized antibody carbohydrate moieties. However, only enzymatically cleavable crosslinkers are disclosed.
Japanese Patent Application J63-57569 discloses bridging agents which are hydrazide compounds of the formula X-SS-A-CONHNH.sub.2, where X is 2-pyridyl or 4-pyridyl and A is a C.sub.1 to C.sub.6 divalent hydrocarbon group (e.g., --CH.sub.2 CH.sub.2 --, --CH.sub.2 CH.sub.2 CH.sub.2 --). These compounds are used to link effector molecules to the sugar chain of an antibody. For example, the antitumor agent methotrexate is linked to an antitumor antibody via the sugar chain to give an antibody/methotrexate complex to be used in target therapy. Linking enzymes with antibodies for enzyme immunoassays is also disclosed.
However, the crosslinkers of J63-57569 react relatively slowly with biomolecules so that they require the antibody to be exposed to the oxidizing medium for a prolonged period of time. The prolonged exposure of the antibody leads to undesirable side reactions which decrease the binding function of the antibody. Further, the crosslinkers of J63-57569 are not sufficiently water soluble to make their handling and manipulation easy.
In addition, the crosslinkers of J63-57569 are achiral and, thus, the possibility of optical isomers does not exist. Since many of the molecules which are to be attached to crosslinkers exist as optically pure isomers, crosslinkers which can exist as optical isomers might possess some advantages, such as site specificity.
Thus, there remains a need for site-specific heterobifunctional crosslinkers which couple to biomolecules or bioaffecting molecules with a high efficiency, react with biomolecules and bioaffecting molecules with a high rate, and possess a high water solubility.